Subtype-Selective Positive Modulation of KCa2.3 Channels Increases Cilia Length

Small-conductance Ca2+-activated potassium (KCa2.x) channels are gated exclusively by intracellular Ca2+. The activation of KCa2.3 channels induces hyperpolarization, which augments Ca2+ signaling in endothelial cells. Cilia are specialized Ca2+ signaling compartments. Here, we identified compound 4 that potentiates human KCa2.3 channels selectively. The subtype selectivity of compound 4 for human KCa2.3 over rat KCa2.2a channels relies on an isoleucine residue in the HA/HB helices. Positive modulation of KCa2.3 channels by compound 4 increased flow-induced Ca2+ signaling and cilia length, while negative modulation by AP14145 reduced flow-induced Ca2+ signaling and cilia length. These findings were corroborated by the increased cilia length due to the expression of Ca2+-hypersensitive KCa2.3_G351D mutant channels and the reduced cilia length resulting from the expression of Ca2+-hyposensitive KCa2.3_I438N channels. Collectively, we were able to associate functions of KCa2.3 channels and cilia, two crucial components in the flow-induced Ca2+ signaling of endothelial cells, with potential implications in vasodilation and ciliopathic hypertension.

In blood vessels, K Ca 2.3 and K Ca 3.1 channel subtypes are often detected on the plasma membrane of endothelial (ET) cells, 3−5 whereas K Ca 2.1 and K Ca 2.2 channel currents are rarely identifiable on the ET cell surface. 6 K Ca 2.3 and K Ca 3.1 channel subtypes seem to have a distinctive distribution and function in ET cells. K Ca 3.1 channels are often found on the ET cell membrane close to the endoplasmic reticulum (ER) Ca 2+ store. 7−9 Ca 2+ release from the ER triggered by acetylcholine or bradykinin receptors may lead to the opening of K Ca 3.1 channels nearby. 10 In contrast, K Ca 2.3 channels seem to colocalize with mechanosensitive or receptor-operated transient receptor potential (TRP) cation channels. 10,11 Ca 2+ influx through these cation channels may activate K Ca 2.3 channels. The outflow of K + can hyperpolarize ET cells, increase the inward electrochemical gradient for Ca 2+ , and augment the Ca 2+ influx, which in turn enhances nitric oxide (NO) releases. 12,13 Non-motile primary cilia are sensory organelles that sense fluid shear stress on the apical membrane of the cells. 14 −16 Fluid flow that produces enough drag force on the top of the cells will bend and activate sensory cilia. Transgenic mouse models with cilia mutations do not survive at birth, confirming the importance of primary cilia in the physiological processes. 17−20 Primary cilia in vasculatures were once thought to be vestigial organelles and nonfunctional remnants. It has since been shown by different laboratories that cilia are mechanosensory organelles. 21−25 Cilia in ET cells sense changes in the fluid shear stress and trigger Ca 2+ signaling and NO releases. 26,27 Primary cilia have been known as specialized Ca 2+ signaling compartments. 28,29 Ca 2+ influx through TRPM4, TRPV4, TRPC1, polycystic kidney disease 2 (PKD2), and L-type voltage-gated Ca 2+ (Ca v ) channels has been considered the main Ca 2+ source for cilia. 28,29 Ca 2+ influx in response to fluid shear stress activates ET K Ca 2.3 channels. 30 In ET cells, K Ca 2.3 channels functionally couple with Ca 2+ -permeable PKD2 11 and TRPV4 31 channels and exert a positive feedback influence on intracellular Ca 2+ signaling. 12,32 However, it is not clear whether this positive feedback mechanism extends back to the cilia, that is, whether the activation of K Ca 2.3 channels increases cilia length. K Ca 2.3 and K Ca 2.2a channels have similar amino acid sequences in their cytoplasmic gates, which makes it difficult to develop subtype-selective positive modulators discriminating these two subtypes. We recently identified the binding site of a prototype K Ca 2.2a/K Ca 2.3 channel modulator, CyPPA. 33 We have synthesized a new series of CyPPA analogues. 34 Here, we report the identification of a subtype-selective K Ca 2.3 channel modulator, compound 4, that is ∼21-fold more potent on potentiating human K Ca 2.3 than rat K Ca 2.2a channels. The subtype selectivity of compound 4 relies on an I-to-V amino acid residue difference between K Ca 2.3 and K Ca 2.2a channels. The pharmacological activation of K Ca 2.3 channels by compound 4 increased cilia length, whereas the pharmacological inhibition of K Ca 2.3 channels by AP14145 decreased cilia length in a cultured ET cell line, suggesting the critical role of K Ca 2.3 channels in the regulation of cilia.

Compound 4 Subtype Selectively Modulates K Ca 2.3 Channels.
A series of CyPPA analogues ( Figure 1A) were synthesized as described in our previous report. 34 The potency of these compounds was measured using inside-out patch clamp electrophysiology recordings with human K Ca 2.3 channels heterologously expressed in HEK293 cells. Positive modulators of K Ca 2 channels require minimal concentration of Ca 2+ to be effective. 35 Therefore, we measured the concentration-dependent responses of the channels to compounds in the presence of 0.15 μM Ca 2+ ( Figure S1). To construct the concentration−response curves, the current amplitudes at −90 mV in response to various concentrations of the compound were normalized to that obtained at the maximal concentration of the compound. The normalized currents were plotted as a function of the compound concentrations. CyPPA, NS13001, and our compounds 2m− 2n, 2p, 2r−2t, 2v, and 4 concentration-dependently potentiated the activity of K Ca 2.3 channels ( Figure 1B). Among them, NS13001 and compounds 2t and 4 exhibited submicromolar EC 50 values ( Figure 1C).  (8), NS13001 (5), 2m (5), 2n (5), 2p (5), 2r (5), 2s (6), 2t (7), 2v (6), and 4 (7). Data are presented as mean ± SD. The responses induced by 10 μM Ca 2+ are considered the maximal currents of the K Ca 2.x channels. 35 To evaluate the efficacy (E max ) of the compounds on K Ca 2.3 channels, the current amplitudes at −90 mV in response to the compounds were normalized to that obtained at 10 μM Ca 2+ [I/I max (%), Figure 1D]. Non-linear regression curve fitting yielded E max values for compounds on K Ca 2.3 channels that are comparable to the E max of CyPPA (96 ± 10%, n = 8, Figure 1E).
The potency of these compounds on potentiating human K Ca 2.3 channels is summarized in Table 1 and compared with their previously determined EC 50 values on rat K Ca 2.2a channels. 34 CyPPA and NS13001 exhibited ∼2.7and ∼4.3fold selectivity for human K Ca 2.3 channels over rat K Ca 2.2a channels (Table 1). Compounds 2t and 4 are ∼6.3 and ∼21 times more potent, respectively, on potentiating the activity of human K Ca 2.3 channels than that of rat K Ca 2.2a channels (Table 1). Among these compounds, compound 4 caught our attention with its ∼21-fold selectivity for human K Ca 2.3 channels over that of rat K Ca 2.2a channels (Table 1). We further evaluated the effects of compound 4 on K Ca 2.1 and K Ca 3.1 channel subtypes. Compound 4 did not potentiate human K Ca 2.1 and human K Ca 3.1 channel subtypes substantively ( Figure S2).

Subtype Selectivity of Compound 4 Relies on the HA/HB Helices.
Our recent study has revealed that the subtype selectivity of CyPPA for K Ca 2.2a and K Ca 2.3 over K Ca 3.1 channels relies on the HA/HB helices. 33 We aligned the amino acid sequences of the rat K Ca 2.2a, human K Ca 2.3, and human K Ca 3.1 channel subtypes in the proximal C terminus (Figure 2A). Rat K Ca 2.2a has a valine residue (V420) equivalent to a methionine residue (M311) of the human K Ca 3.1 channel in the HA helix. In the HB helix, rat K Ca 2.2a has a lysine residue (K467), corresponding to an arginine residue (R355) of the human K Ca 3.1 channel. The V-to-M and K-to-R discrepancies between the amino acid sequences of rat K Ca 2.2a and human K Ca 3.1 channels provide an explanation for the subtype selectivity of CyPPA. 33 We then set out to explore the structural determinants for the ∼21-fold subtype selectivity of compound 4 for human K Ca 2.3 over rat K Ca 2.2 channels. Human K Ca 2.3 has an isoleucine (I568) equivalent to V420 in the HA helix of rat K Ca 2.2a channels (Figure 2A). The side chain of K Ca 2.3_I568 would be bulkier than that of K Ca 2.2a_V420. Thus, the different sizes of a valine (rat K Ca 2.2a_V420) and an isoleucine (human K Ca 2.3_I568) may constitute the structural determinants for the subtype selectivity of compound 4. We tested this hypothesis by mutating K Ca 2.3_I568 to its corresponding amino acid residue in K Ca 2.2a, a valine ( Figure 2B). The K Ca 2.3_I568V mutant channel exhibited an EC 50 value of 6.2 ± 1.3 μM (n = 6), which is ∼20-fold less sensitive to compound 4 than the K Ca 2.3_WT with an EC 50 value of 0.31 ± 0.07 μM (n = 7, Figure 2C). The K Ca 2.3_I568V mutation did not affect the E max values to compound 4, compared with the K Ca 2.3_WT channel ( Figure 2D,E). The K Ca 2.3_I568V mutation did not influence the apparent Ca 2+ sensitivity of K Ca 2.3 channels ( Figure S3A,B).
The corresponding mutation in rat K Ca 2.2a channels (K Ca 2.2a_V420I) did not change either the apparent Ca 2+ sensitivity of K Ca 2.2a channels ( Figure S4A,B) or the E max to compound 4 ( Figure S4C,D). The K Ca 2.2a_V420I increased the sensitivity of the channel to compound 4 ( Figure S4E,F), corroborating the results acquired from the corresponding K Ca 2.3_I568V mutation ( Figure 2B,C).

Pharmacological Modulation of K Ca 2.3 Channels Affected Cilia Length.
Recently, we identified K Ca 2.3 channels as the predominant subtype expressed in a mouse ET cell line, whereas the expression of K Ca 2.1, K Ca 2.2, and K Ca 3.1 channel subtypes was not detected by immunoblots. 36 Thus, we examined whether negative modulation by AP14145 of K Ca 2.3 channels affected the cilia length of the ET cells. AP14145 inhibited K Ca 2.3 channels with an IC 50 value of 0.97 ± 0.39 μM (n = 5, Figure S5).
ET cells were incubated with AP14145 (20 μM) for 2 days before cells reached confluency, and the cilia length was evaluated using immunostaining with the antibody of the ciliary marker acetylated α-tubulin (green) and the nuclear marker DAPI (blue, Figure S6A). AP14145 shortened cilia to 2.8 ± 0.1 μm, compared with 6.3 ± 0.3 μm of the solvent control group ( Figure S6B,C), suggesting a regulatory role of K Ca 2.3 channels in the cilia length of ET cells.
Compound 4 potentiated K Ca 2.3 channels with an EC 50 value of 0.31 ± 0.07 μM (n = 7) (Table 1 and Figure 1C). ET cells were incubated with compound 4 (20 μM) for 2 days before cells reached confluency, and the cilia length was evaluated using immunostaining with the antibody of the ciliary marker acetylated α-tubulin (green) and the nuclear marker DAPI (blue, Figure 3A). Compound 4 increased the cilia length to 6.1 ± 0.6 μm compared with 4.3 ± 0.3 μm of the solvent control group ( Figure 3B,C), suggesting potential therapeutic usefulness of K Ca 2.3 channel positive modulators (e.g., compound 4) in ciliopathy disease states with abnormal cilia.
To confirm the elongating effect of compound 4 on cilia ( Figure 3), an additional ciliary marker Arl13b was used to measure the cilia length ( Figure S7A). Also, the γ-tubulin was used as a marker for the basal body (base of a cilium), which 0.64 ± 0.12 34 0.60 ± 0.10 34 2r 3.0 ± 0.7 34 2    sensitivity of the channels, 38 whereas negative modulators decrease the apparent Ca 2+ sensitivity of the channels. 39 To rule out the possibility that compound 4 and AP14145 affected cilia length through their off-target effects other than K Ca 2.3 channels, we heterologously expressed mutant K Ca 2.3 channels with altered apparent Ca 2+ sensitivity ( Figure 4). When expressed in ET cells, the K Ca 2.3 channels exhibited an apparent Ca 2+ sensitivity of 0.67 ± 0.11 μM (n = 6). The G351D mutation significantly increased the apparent Ca 2+ sensitivity to 0.16 ± 0.04 μM (n = 7), while the I438N mutation significantly reduced the apparent Ca 2+ sensitivity to 1.8 ± 0.3 μM (n = 5, Figure 4). Immunoblots (Figures S8A−  The higher the apparent Ca 2+ sensitivity of the mutant channel, the more likely the K Ca 2.3 channel is opening and then augmenting the Ca 2+ influx in a positive feedback mechanism. The overexpression of K Ca 2.3_WT led to a slightly increased cilia length (6.3 ± 0.2 μm) compared with the control (5.3 ± 0.5 μm, Figure 5). K Ca 2.3_G351D mutant channels with hypersensitivity to Ca 2+ increased the cilia length even more drastically (15.3 ± 0.7 μm), while the K Ca 2.3_I438N mutant channels with hyposensitivity to Ca 2+ reduced the cilia length (2.2 ± 0.3 μm, Figure 5), confirming a role of the K Ca 2.3 channel in the regulation of cilia length.

Pharmacological Intervention of K Ca 2.3 Channels Affected Ca 2+
Signaling. The opening of K Ca 2.3 channels induces hyperpolarization, which may increase the inward electrochemical gradient for Ca 2+ and thus augment the Ca 2+ influx. Next, we investigated whether the positive modulation or negative modulation of K Ca 2.3 channels affected the Ca 2+ signaling, using fluorescence Ca 2+ imaging ( Figure 6). Flowinduced cytosolic Ca 2+ transients were measured using a ratiometric, high-affinity intracellular Ca 2+ indicator Fura-2AM. Compared with the control ET cells (Figure 6A), the AP14145-treated ET cells exhibited much weaker Ca 2+ transients ( Figure 6B). In contrast, the compound 4-treated ET cells exhibited more prominent Ca 2+ transients ( Figure  6C) than the control cells. The significant effects of a negative modulator AP14145 and a positive modulator compound 4 on the flow-induced peak Ca 2+ values ( Figure 6D) suggest a link between the K Ca 2.3 channel opening and Ca 2+ signaling, triggered by the shear stress. We have previously generated the non-ciliated IFT88 knockout (KO) mouse ET cells. 40 Using these cells, we further validate that flow-induced cytosolic Ca 2+ transients were largely abolished in IFT88 KO ET cells, suggesting the essential role of cilia in flow-induced Ca 2+ signaling ( Figure S9).

DISCUSSION
Among the four channel subtypes encoded by the mammalian KCNN genes, K Ca 2.3 closely resembles the K Ca 2.2 channel subtype in pharmacology. 41 The human K Ca 2.2a channel does not express as well as the rat K Ca 2.2a channel, which prevented us from performing inside-out patch clamp experiments. Human and rat K Ca 2.2a channels are highly homologous, with differences only in the distal cytoplasmic N-and Ctermini. In the transmembrane domains and in the cytoplasmic gate including the HA/HB helices (highlighted in green), which CyPPA interacts with, the similarity is 100% ( Figure  S10). The prototype subtype-selective positive modulator, CyPPA achieved selectivity for K Ca 2.2 and K Ca 2.3 channels over K Ca 2.1 and K Ca 3.1 subtypes. 35 CyPPA is also ∼2.7 times more potent on human K Ca 2.3 than on rat K Ca 2.2a channels (Table 1). In this study, we identified a positive modulator, compound 4, that is ∼21-fold selective for human K Ca 2.3 over rat K Ca 2.2a channels (Table 1). Compound 4 is largely inactive on human K Ca 2.1 and human K Ca 3.1 channels ( Figure S2). The significance of this study is not limited to compound 4 itself with an EC 50 of ∼0.3 μM and a modest subtypeselectivity for human K Ca 2.3 over rat K Ca 2.2a channels. The subtype selectivity of compound 4 for human K Ca 2.3 over rat K Ca 2.2a channels relies on an I-to-V discrepancy in the HA/ HB helices between the two subtypes (Figures 2 and S4), which may offer an opportunity for the development of even more subtype-selective modulators.
The expression of K Ca 2.3 together with K Ca 3.1 channels on the plasma membrane of ET cells is well-documented. 3−5 K Ca 2.3 channels functionally couple with mechanosensitive and TRP Ca 2+ -entry channels (e.g. PKD2 11 and TRPV4 31 ). We observed a positive feedback effect of K Ca 2.3 channels on the flow-induced intracellular Ca 2+ signaling through cilia ( Figure  6). Most importantly, the positive feedback extends back to cilia themselves as the positive modulator compound 4 increased the cilia length (Figure 3), while the negative modulator AP14145 reduced the cilia length ( Figure S6). These observations allow us to connect K Ca 2.3 channels and cilia, two crucial components in the flow-induced Ca 2+ signaling in ET cells, with implications in vasodilation and blood pressure regulation.
The regulation of cilia length by K Ca 2.3 channel positive and negative modulators (Figures 3 and S6) has been corroborated by the effects on cilial length of the mutant K Ca 2.3 channels with altered apparent Ca 2+ sensitivity (Figures 4 and 5). Expression of the Ca 2+ -hypersensitive K Ca 2.3_G351D mutant channel increased the cilia length, while the Ca 2+ -hyposensitive K Ca 2.3_I438N mutant channel reduced the cilia length ( Figure  5). It is noteworthy that the mouse K Ca 2.3_G351D mutation used in our study is equivalent to the human K Ca 2.3_G350D mutation, which causes Zimmermann-Laband syndrome (ZLS). 42 It has been speculated that during human embryonic development, excessive hyperpolarization due to hypersensitivity to Ca 2+ of the ZLS-related mutant K Ca 2.3 channels might result in exaggerated vasodilation in response to shear stress. This in turn might cause edema and vascular ruptures in critical phases of embryonic development, leading to distal digital hypoplasia with aplastic or hypoplastic nails and terminal phalanges. 42 Our results showed that the equivalent mouse K Ca 2.3_G351D mutation caused hypersensitivity to Ca 2+ (Figure 4), which may contribute to vasodilation mediated by the endothelium-derived hyperpolarization. 8,43,44 Our finding here that the expression of K Ca 2.3_G351D mutant channels increased cilia length in ET cells ( Figure 5) could also be translated into increased sensitivity and vasodilation in response to blood flow. Both of these two mechanisms might underlie the vasodilation and vascular rupture speculated in the embryonic development of ZLS patients, although further studies will be needed to elucidate the developmental biology.
We and other laboratories have previously reported that rapamycin increases cilia length in epithelial cells, resulting in the inhibition of cell proliferation. 45,46 On the other hand, rapamycin-induced cilia length increase correlates to an elevated response to fluid shear stress in ET cells. 47 The function of primary cilia as mechanosensory organelles depends on the length of cilia; lengthening primary cilia enhance cellular mechanosensitivity. 48,49 Dopamine, for example, also increases cilia length and function, resulting in enhanced cellular mechanosensitivity. 50 While dopamine specificity was a concern, drugs that improve sensory cilia function by elongating cilia length have been coined "ciliotherapy". 51 A more specific cilia-targeted therapy in ET cells has also been proposed to remedy hypertension. 52,53 We therefore are hopeful that subtype-selective positive modulators of KCa2.3 channels (e.g., compound 4) would have a great potential to be a potential ciliotherapy. Table 2.

Electrophysiology.
The effect of compounds on the K Ca 2.x/ K Ca 3.1 channels was investigated as previously described. 54,55 Briefly, the rat K Ca 2.2a, human K Ca 2.1, human K Ca 2.3, or human K Ca 3.1 channel cDNA constructs were either generated in-house or through molecular cloning services (Genscript, Piscataway, NJ, USA). These channel cDNAs in the pIRES2-AcGFP1 vector, along with calmodulin cDNA in the pcDNA3.1 + vector, at a ratio of 10:1 (ORF ratios), were transfected into cells using the calcium−phosphate method. K Ca currents were recorded 1−2 days after transfection using an Axon200B amplifier (Molecular Devices, San Jose, CA) at room temperature. The resistance of the patch electrodes ranged from 3 to 5 MΩ. The pipette solution contained the following (in mM): 140 KCl, 10 Hepes (pH 7.4), and 1 MgSO 4 . The bath solution containing (in mM) 140 KCl, 10 Hepes (pH 7.2), 1 EGTA, 0.1 Dibromo-BAPTA, and 1 HEDTA was mixed with Ca 2+ to obtain the desired free Ca 2+ concentrations, calculated using the software written by Chris Patton (https://somapp.ucdmc.ucdavis.edu/pharmacology/ bers/maxchelator/webmaxc/webmaxcS.htm). The Ca 2+ concentra- High-resistance seals (>1 GΩ) were formed before inside-out patches were obtained. The seal resistance of inside-out patches was >1 GΩ, when the intracellular face was initially exposed to a zero-Ca 2+ bath solution. Currents were recorded by repetitive 1-s-voltage ramps from −100 to +100 mV from a holding potential of 0 mV. The currents were filtered at 2 kHz and digitized at a sampling frequency of 10 kHz. At the end of the experiment, the integrity of the patch was examined by switching the bath solution back to the zero-Ca 2+ buffer. Data from patches, which maintained the seal resistance (>1 GΩ) after solution changes, were used for further analysis.
To measure the effect of the positive modulators, the intracellular face was exposed to bath solutions with 0.15 μM Ca 2+ . One minute after the switching of bath solutions, 10 sweeps with a 1 s interval were recorded at a series of concentrations of the compound in the presence of 0.15 μM Ca 2+ . The maximal K Ca 2.x/K Ca 3.1 current in response to 10 μM Ca 2+ was then recorded.
To measure the effect of the negative modulator Ap14145, the intracellular face was exposed to bath solutions with 0.5 μM Ca 2+ . One minute after the switching of bath solutions, 10 sweeps with a 1 s interval were recorded at a series of concentrations of AP14145 in the presence of 0.5 μM Ca 2+ .

Cilia Measurements.
Cilia length was measured by direct immunofluorescence for the cilia marker with anti-acetylated αtubulin or Arl13b staining. The cells were fixed for 10 min (4% paraformaldehyde/2% sucrose in PBS) and permeabilized for 5 min (10% Triton X-100). Acetylated α-tubulin (1:10,000 dilution, Sigma-Aldrich, St. Louis, MO) or Arl13b (1:50 dilution, Proteintech, Rosemont, IL) and fluorescein isothiocyanate-conjugated (1:1000 dilution, Vector Labs Burlingame, CA) antibodies were each incubated with the cells for 1 h at 37°C. Microscope slides were then mounted with DAPI (Southern Biotech, Birmingham, AL) hard set mounting media. A Nikon Eclipse Ti-E inverted microscope with NIS-Elements imaging software (version 4.30) was used to capture the images of primary cilia. Automated image acquisition was conducted in 100× magnification fields. Cilia length analysis followed a standard calculation as previously described. 56 4.4. Flow-Induced Ca 2+ Measurements. Cells were loaded with 5 μM Fura2-AM (Thermo Fisher Scientific, Waltham, MA) at 37°C for 30 min. Cells were then washed with Dulbecco's phosphatebuffered saline and observed under a 40× objective lens using a Nikon Eclipse Ti-E microscope controlled by Elements software. Cytosolic calcium was observed by recording Ca 2+ -bound Fura excitation fluorescence at 340/380 nm and emission at 510 nm. Baseline Ca 2+ was observed for 5 min prior to data acquisition. Fluid shear stress was then applied to cells utilizing an Instech P720 peristaltic pump with an inlet and outlet setup. The fluid was perfused on the glassbottom plates at a shear stress of 5 dyn/cm 2 . After each experiment, the maximum calcium signal was obtained with ATP (10 μM) to confirm cell viability. Conditions for all experiments were maintained at 37°C and 5% CO 2 in a stage top cage incubator (okoLab, Burlingame, CA). Ca 2+ analysis followed a standard calculation as previously described. 56 4.5. Immunoblots. The protein concentrations in ET cell lysates were determined using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). Equal amounts of protein (15 μg) were separated by sodium dodecyl sulfate−polyacrylamide gel electrophoresis gel (Bio-Rad Laboratories, Hercules, CA). The proteins were transferred to polyvinylidene fluoride (PVDF) membranes and incubated overnight at 4°C with the primary GFP antibody (1:2000; Novus Biological, Centennial, CO) or GAPDH antibody (1:5000; Abcam, Waltham, MA). The PVDF membranes were washed with Tris-buffered saline (0.1% Tween 20) and incubated with the anti-rabbit antibody (1:3000; cell signaling technology, Danvers, MA) as the secondary antibody for 1 h at room temperature and then washed with Tris-buffered saline (0.1% Tween 20). The chemiluminescence signals were detected on a ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA) after incubation with Luminol/enhancer solution (Thermo Fisher Scientific, Waltham, MA). Densitometry analyses were performed using the ImageJ computer program. 4.6. Data and Statistical Analysis. Patch clamp recordings were analyzed using Clampfit 10.5 (Molecular Devices LLC, San Jose, CA), and concentration−response curves were analyzed in GraphPad Prism 9.0.2 (GraphPad Software Inc., La Jolla, CA). To construct the concentration-dependent potentiation of channel activities by the compound, the current amplitudes at −90 mV in response to various concentrations of the compound were normalized to that obtained at a maximal concentration of the compound. The normalized currents were plotted as a function of the concentrations of the compound. EC 50 values and Hill coefficients were determined by fitting the data points to a standard concentration−response curve [Y = 100/(1 + (X/EC50)̂− Hill)]. To assess the efficacy of the compound, the current amplitudes obtained at the maximal concentration of the compound were normalized to the maximal K Ca 2.x/K Ca 3.1 current in response to 10 μM Ca 2+ . Concentration−response curves were acquired from multiple patches for each data set. Each curve was fitted individually, which yielded the EC 50 value for that curve. EC 50 values are shown as mean ± SD obtained from multiple patches, and the number of patches is indicated by n.
The Student's t-test was used for data comparison if there were only two groups. One-way ANOVA and Tukey's post hoc tests were used for data comparison of three or more groups. Post hoc tests were carried out only if F was significant and there was no variance in homogeneity.